Methods and articles of manufacture for the treatment of animals

ABSTRACT

Methods are disclosed for the collection and processing of amniotic material in animals. These methods involve collection of amniotic material directly during parturition or caesarian section in animals for the processing of regenerative wound treatments and tissue repairs without culturing or utilization of any excess manipulation of tissue.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application62/234,354, filed Sep. 29, 2015, entitled “Methods and Articles ofManufacture for the Treatment of Animals, the contents of which areincorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERAL SPONSORSHIP

Inventions described herein were not conceived or reduced to practicewith Federal sponsorship.

FIELD OF THE INVENTION

The present disclosure provides processed fetal tissues and cellssuitable for promoting healing and methods of using these fetal tissuesand cells to promote healing at the site of an injury.

BACKGROUND OF THE INVENTION

Wounds and injuries in animals and humans can sometimes be difficult totreat. Failure to adequately treat and promote healing can place theanimal or individual in jeopardy with potential loss of function in aneffected limb or amputation, or even loss of life.

It would be useful to have methods and articles of manufacture thatpromote healing in animals and humans. As used herein, unless thecontext requires otherwise, the term “animal: encompasses and includeshumans.

SUMMARY OF THE INVENTION

Embodiments of the present invention feature methods and articles ofmanufacture that promote healing in animals.

The fetal tissue and cellular compositions disclosed herein have manyadvantages for use in promoting healing of injuries including immuneprivilege, an absence of associated ethical issues, and no requirementof invasive procedures for harvesting the cells and tissues. Inaddition, the treatment regimen disclosed herein is remarkably effectiveat promoting the rapid healing of open wounds within one to two weeks.

One embodiment is directed to an article of manufacture. The articlecomprises a dried particulate mixture of mechanically decellularizedamnion obtained from one or more animals compatible with a subjectanimal. The dried particulate mixture is capable of reconstitution toform a reconstituted medicament for administration to the subject animalto promote healing.

By way of example, without limitation, a therapeutically effectiveamount of the reconstituted medicament is applied by injecting at oraround the periphery of an internal or external injury, or applied tothe subject animal by way of dropper or spray, used as a wash, powder ordressing for wounds, surgically implanted, or the like to promotehealing. The treatment may also be applied by way of nebulization ororal. For example, without limitation, applications to an eye of asubject animal feature drops.

In one aspect, one milliliter of reconstituted medicament is the amountof particulate matter obtained from the mechanically decellularizedamnion of about 1.5 10⁻² cm³ to 5 10⁻² cm³ of amnion. In one aspect, themechanically decellularized amnion is filtered to contain particles ofless than about 100 microns in diameter. In one aspect, particulatesuspension containing particles of less than about 20-100 microns indiameter is well suited for the treatment of the eye injury.

Embodiments of the present invention feature a particulate mixturecomprising particles. The particles have an approximate diameter of lessthan 500μ, or less than 400μ, or less than 300μ, or less than 200μ, orless than 150μ, or less than 100μ, or less 90μ, or less than 80μ, orless than 70μ, or less than 60μ, or less than 50μ, or less than 40μ, orless than 30μ, or less than 20μ, or less than 10μ. In other embodiments,the particles have a diameter of more than 10μ, or more than 20μ, ormore than 30μ, or more than 40μ, or more than 50μ, or more than 60μ, ormore than 70μ, or more than 80μ, or more than 90μ, or more than 100μ, ormore than 200μ, or more than 300μ, or more than 400μ, or more than 500μ.In other embodiments, the particles have an approximate diameter ofabout 500μ, or about 400μ, or about 300μ, or about 200μ, or about 150μ,or about 100μ, or about 50μ, or about 25μ.

In certain embodiments, the article comprises a dried particulatemixture of mechanically decellularized amnion obtained from one or moreanimals compatible with a subject animal, and fetal cells obtained fromone or more animals compatible with a subject animal to form aparticulate cellular suspension medicament. The particulate cellularsuspension medicament is administered to a subject animal to promotehealing.

By way of example, without limitation, a therapeutically effectiveamount of the particulate cellular suspension medicament can be injectedaround the periphery of an internal or external injury to promotehealing, applied subject animal by way of dropper or spray, used as awash, powder or dressing for wounds, surgically implanted, or the like.For example, without limitation, applications to an eye of a subjectanimal feature drops.

A therapeutically effective amount of the particulate cellularsuspension medicament comprises about 10¹ to 10²⁰ cells per mL ofmedicament. Other embodiments feature about 10³ to about 10⁷ cells permL.

A further embodiment of the article further comprises a fetal tissuewrap. The tissue wrap can comprise amnion tissue obtained from one ormore animals compatible with a subject animal. The wrap is constructedand arranged for placement in juxtaposition with a site of injury topromote healing.

The wrap, comprising fetal tissue, can be air-dried for about 1 minuteto about 48 or more. In another embodiment the wrap is air-dried forabout 1 hour to about 12 hours. In another embodiment, the wrap isair-dried for about 1 hour to about 6 hours. In another embodiment, thewrap is air-dried for about 1 hour to about 3 hours. In anotherembodiment, the wrap is air-dried for about 1 hour to about 2 hours.

In another aspect of the invention, one embodiment features a kit forthe treatment of a subject animal. One kit comprises a dried particulatemixture of mechanically decellularized amnion obtained from one or moreanimals compatible with a subject animal that can be capable ofreconstitution to form a reconstituted medicament.

A further embodiment of the kit comprises fetal cells obtained from oneor more animals compatible with a subject animal that are compatiblewith a particulate mixture obtained from the mechanicaldecellularization of amnion isolated from one or more animals. The fetalcells are applied in cooperation or concurrently with the reconstitutedmedicament or form a combined medicament comprising a particulatecellular suspension and/or amniotic liquid medicament for administrationto the subject animal to promote healing.

A further embodiment of the kit comprises a tissue wrap obtained fromone or more animals compatible with a subject animal. The tissue wrapcomprises amnion tissue constructed and arranged for placement injuxtaposition with the site of injury. The tissue wrap is applied incooperation or concurrently with the reconstituted medicament or acombined medicament comprising a particulate cellular or acellularsuspension medicament with or without mechanically decellularized amnionand/or amniotic liquid for administration to the subject animal topromote healing.

A further embodiment of the present invention is directed to a method ofpromoting healing in a subject animal. One embodiment of the presentmethod features the steps of administering to the site of an injury atleast one of the group consisting of a reconstituted medicament, areconstituted amnion suspension with or without cells and a tissue wrapall of which have been previously described.

For example, without limitation, in one embodiment, a method comprisesthe step of applying a reconstituted medicament. The reconstitutedmedicament is made from a dried particulate mixture of mechanicallydecellularized amnion obtained from one or more animals compatible witha subject animal.

Another method features the step of applying, by way of injection, aparticulate cellular suspension medicament to the periphery of theinjury. The particulate cellular suspension medicament suspensioncomprises particles derived from the mechanical decellularization ofamnion obtained from one or more animals compatible with the subjectanimal, and isolated amniotic fluid cells obtained from the one or moreanimals compatible with the subject animal.

In one aspect, the method further comprises the step of applying atissue wrap to the site of the injury. The wrap comprises amnion tissuecompatible with the subject animal constructed and arranged forplacement in juxtaposition with the site of injury. The amnion tissue isair-dried for about 1 minute to about 48 hours or more. In anotherembodiment the amnion tissue is air-dried for about 1 hour to about 12hours. In another embodiment, the amnion tissue is air-dried for about 1hour to about 6 hours. In another embodiment, the amnion tissue isair-dried for about 1 hour to about 3 hours. In another embodiment, theamnion tissue is air-dried for about 1 hour to about 2 hours.

In one aspect of the method, the site of injury is debrided prior toapplying one or more of the group consisting of a cellular reconstitutedsuspension, or a reconstituted acellular suspension medicament and atissue wrap.

A further embodiment is directed to a method of making a driedparticulate mixture of mechanically decellularized obtained from one ormore animals compatible with a subject animal. The dried particulatemixture is capable of reconstitution to form a reconstituted medicamentfor administration to the subject animal to promote healing. The methodcomprises the step of mechanically decellularizing amnion tissue to formparticles capable of reconstitution.

A further embodiment is directed to a method of making a particulatecellular suspension medicament. The method comprises the steps ofproviding a dried particulate mixture of mechanically decellularizedamnion obtained from one or more animals compatible with a subjectanimal, and fetal cells obtained from one or more animals compatiblewith a subject animal to form a particulate cellular suspensionmedicament and forming a particulate cellular suspension. Theparticulate cellular suspension medicament is administered to a subjectanimal to promote healing.

A further embodiment of the present invention features methods of makinga tissue wrap, a particulate mixture medicament and a particulatecellular suspension medicament. One embodiment of the method of makingthe tissue wrap comprises the steps of applying amnion tissue to asupport to form a supported amnion. The supported amnion is next airdried to form the tissue wrap which is placed in a suitable containmentmeans until applied to a site of injury.

These and other features and advantages will be apparent upon viewingthe Figures that are briefly described below and upon reading thedetailed description that follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a kit embodying features of the present invention;

FIG. 2 depicts a tissue wrap embodying features of the presentinvention; and,

FIG. 3 shows a tissue wrap embodying features of the present inventionin a container.

DETAILED DESCRIPTION

Unless otherwise defined herein, scientific and technical terms usedherein have the meanings that are commonly understood by those ofordinary skill in the art. In the event of any latent ambiguity,definitions provided herein take precedence over any dictionary orextrinsic definition. Unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular. The use of “or” means “and/or” unless stated otherwise. Theuse of the term “including”, as well as other forms, such as “includes”and “included”, is not limiting.

It is noted here that as used in this specification and the appendedclaims, the singular forms “a”, “an”, and “the” also include pluralreference, unless the context clearly dictates otherwise.

The term “about” or “approximately” means within 10%, and morepreferably within 5% (or 1% or less) of a given value or range.

As used herein, the term “isolated cell” refers to a cell that has beenremoved from its in-vivo location.

As used herein, the term “decellularization” refers to a process thatremoves cells from a tissue while preserving the native ultrastructureand composition of the extracellular matrix (ECM). For example, anamnion particulate mixture can be obtained by decellularizing a fetaltissue comprising amnion.

There are a number of methods of decellularization of tissue known inthe art, including, but not limited to, chemical agents, hypotonic andhypertonic solutions, detergents (e.g., Triton-X), alcohols, solvents(e.g., tributyl phosphate (TBP), biologic agents (e.g., collagenase,trypsin, lipase, nucleases, α-galactosidase), non-enzymatic agents(e.g., chelating agents such as EDTA or EGTA), physical agents (e.g.,temperature, force and pressure, non-thermal irreversible, mechanical,electroporation (NTIRE) (see, for example, Crapo et al., Biomaterials.2011; 32(12): 3233-3243). In certain embodiments, one or a combinationof the aforementioned methods may be used to decellularize a tissue.However, methods that preserve the complex composition andthree-dimensional ultrastructure of the extracellular matrix (ECM) arepreferred.

In one embodiment, a tissue is mechanically decellularized, e.g., bycryofractionation, a procedure in which a tissue is frozen and ground ina cryomill to produce a mixture of particles. Such particles areobtained from the cryofractionation of about 0.5 cm², or about 1 cm², orabout 1.5 cm², or about 2 cm², or about 2.5 cm², or about 3 cm², orabout 3.5 cm² or about 4 cm², or about 4.5 cm² to about 5 cm² of amnionor more. The amnion can have a thickness of from about 500μ to 50μ, orfrom 400μ to about 50μ, or from about 300μ to 50μ, or from about 200μ toabout 50μ, or from about 150μ to about 50μ, from about 100μ to about 50μor from about 50μ to about 25μ or less. In another embodiment, theamnion has a thickness of about 500μ, or about 400μ, or about 300μ, orabout 200μ, or about 150μ, or about 100μ, or about 50μ, or about 25μ orless.

As used herein, 1μ refers to a micrometer or 10⁻⁶ meters.

The term “amnion” refers to a thin, cellular, extra-embryonic membranethat forms the inner membrane of a closed sac surrounding and protectingan embryo in reptiles, birds, and mammals. The sac contains the fetusand amniotic fluid, in which the embryo is immersed, nourished andprotected. Typically, the amnion is a tough, transparent, nerve-free,and nonvascular membrane consisting of two layers of cells: an inner,single-cell-thick layer of ectodermal epithelium and an outer coveringof mesodermal, connective, and specialized smooth muscular tissue. Inthe later stages of pregnancy, the amnion expands to come in contactwith the inner wall of the chorion creating the appearance of a thinwall of the sac extending from the margin of the placenta. The amnionand chorion are closely applied, though not fused, to one another and tothe wall of the uterus. Thus, at the later stage of gestation, the fetalmembranes are composed of two principal layers: the outer chorion thatis in contact with maternal cells and the inner amnion that is bathed byamniotic fluid. The amnion has multiple functions, e.g., as a coveringepithelium, as an active secretary epithelium, and for intenseintercellular and transcellular transport.

As used herein, the term “tissue” refers to an aggregate of similarcells and associated extracellular matrix (ECM) forming a definite kindof organized material with a specific function, in a multicellularorganism.

As used herein, an “amnion tissue” refers to the isolated cellular,extra-embryonic amnion membrane that is detached from the chorion. Inone embodiment, the amnion tissue is air-dried. In another embodiment,the amnion is air-dried for about 60 to about 90 minutes or more atambient temperature (i.e. about 18 to 24° C.).

As used herein, a “particulate mixture” refers to the powder orparticles obtained from the cryofractionation of amnion.

As used herein, the term “fetal tissue” refers to extra-embryonictissues including, but not limited to, amnion, chorion, yolk sac, theallantois, umbilical cord and/or fetal placenta (villous chorion).

As used herein, the term “fetal cells” refers to cells resident in theextra-embryonic tissues including, but not limited to, amnion, chorion,yolk sac, the allantois, umbilical cord, fetal placenta (villouschorion) and/or amniotic fluid. In certain embodiments, the term “fetalcells” refer to isolated fetal cells.

In certain embodiments, the term “fetal cells” refers to unfractionatedcells of the amniotic fluid including epithelial and/or amniotic fluidor membrane-derived mesenchymal stem cells (see U.S. Patent PublicationNo. US 2013/0230924, which is incorporated by reference herein in itsentirety).

The term “injury” means a pathological condition, such as, by way ofexample, without limitation, a wound, incision, a break in the skin,bone, tendon, ligament, muscle, neoplasia, eye, and soft tissues, aninflammation, infection, or other disease condition.

The term “promoting healing” refers to causing a favorable resultcompared to no treatment. The favorable result comprises any one or moreof the following such as reduction of scarring, reduction ofinflammation, regrowth of normal tissue or growth of scar tissue,improved load bearing on a limb movement, closure of wound, reduction ininfection and reduction in mortality associated with the underlyingpathology.

The term “compatible with a subject animal” denotes the origin of thetissue as being from the same species or closely related species.

In other embodiments, the term “compatible with a subject animal” refersto an xenograft, i.e., a tissue graft from different species.

In another embodiment, the term “compatible with a subject animal”refers to allografts, i.e., a tissue from one individual to another ofthe same species with a different genotype.

As used herein, an “animal” refers to living multi-cellular vertebrateorganisms, a category that includes, for example, mammals, birds,reptiles, and amphibians. The term mammal includes both human andnon-human mammals. Similarly, the term “subject animal” includes bothhuman and non-human subjects. The term “subject animal” refers to anindividual human or animal which has a site of injury or disease.

As used herein, a non-human animal can refer to a mammal including, butnot limited to, a domesticated animal such as a dog, a racing dog,sheep, a pig, a goat, cattle, a zebu, a cat, a guinea pig, a donkey,water buffalo, including “river buffalo” and “swamp buffalo”, a horse, aracing horse, a dromedary camel, a yak, a bactrian camel, a llama, analpaca, a ferret, a mouse, a bali cattle, a gayal, a rabbit, a rat and alab rat, a silver fox or a hedgehog.

In certain embodiments, a non-human animal can refer to mammals kept inzoos including, but not limited to, zebra, gazelle, wolves, wild swine(pigs & hogs), wild cattle, warthogs, vervet monkeys, two-toed sloths,tree pangolins, tigers, tapirs, tamandua or lesser anteaters, takins,sun bears, striped hyena, spotted hyena, spiral-horned antelope, somaliwild ass, snow leopards, small cats, sloth bears, singing dogs, siamang,serval, sea lions, rock hyrax, rhinoceros, reindeer, red pandas, pygmymarmosets, pygmy hippopotamus, przewalski's horses, pronghorns, prairiedogs, porcupines, polar bears, painted dogs, otters, oryx, orangutan,okapi, ocelot, nubian ibex, nile lechwe, naked mole-rats, mountain lions(puma, cougar), monkeys, meerkat, mangabey, mandrill, lynx and bobcats,lions, leopards, lemur, jaguars, honey badgers (ratel), hippos,hamadryas baboons, guenon, guanaco, gorillas, giraffe, giant pandas,giant anteaters, gelada baboons, fossa, fishing cats, elephants,echidna, dhole, coquerel's sifaka, clouded leopards, chimpanzees,cheetahs, tigers, caracals, capybara, camels, brown bears, bonobos,binturongs, bat-eared fox, bats, armadillos, antelope, andean(spectacled) bears and agouti.

In certain other embodiments, a non-human animal can refer to mammalsconsidered by the World Wildlife Fund to be endangered including, butnot limited to, the amur leopard, black rhino, cross river gorilla,javan rhino, mountain gorilla, pangolin, saola, south china tiger,sumatran elephant, sumatran orangutan, sumatran rhino, sumatran tiger,vaquita, western lowland gorilla, yangtze finless porpoise, african wilddog, amur tiger, asian elephant, bengal tiger, black spider monkey,black-footed ferret, blue whale, bonobo, bornean orangutan, borneo pygmyelephant, chimpanzee, eastern lowland gorilla, fin whale, ganges riverdolphin, giant panda, hector's dolphin, indian elephant, indochinesetiger, indus river dolphin, malayan tiger, north atlantic right whale,orangutan, sea lions, sei whale, snow leopard, Sri Lankan elephant,tigers and whales.

In certain embodiments, a non-human animal can refer to marsupials,including, but not limited to, wallabies, koalas, possums, opossums,kangaroos, bandicoots, wombats, bettongs, bilbys, quolls, quokkas andthe Tasmanian devil.

The term “reconstituted” means that that an aqueous liquid is added tomake the medicament. A liquid for reconstitution comprises abiocompatible solution such as normal saline, e.g. phosphate bufferedsaline (PBS) or amniotic fluid. A preferred liquid for reconstitution iscalcium-free sterile, non-pyrogenic isotonic solution suitable forintravenous administration. For example, without limitation, one suchliquid is sold under the trademark PlasmaLyte A™ in a single dosecontainer for intravenous administration. Each 100 mL contains 526 mg ofSodium Chloride, USP (NaCl); 502 mg of Sodium Gluconate (C₆H₁₁NaO₇); 368mg of Sodium Acetate Trihydrate, USP (C₂H₃NaO₂.3H₂O); 37 mg of PotassiumChloride, USP (KCl); and 30 mg of Magnesium Chloride, USP (MgCl₂.6H₂O).It contains no antimicrobial agents. The pH is 7.4.

The term “administering” means applying or injecting or ingesting themedicament. The term “applying” is used broadly and includes uses suchas washes and implantation.

Connective soft tissue defects or injuries often occur by damage to theextracellular matrix (ECM) that forms muscles, ligaments or tendons inmammals. Collagen is the most abundant structural protein in theconnective tissue (ECM) and acts as a natural scaffold for cellularattachment in the body.

Amnion is an abundant source of collagen, as well as the other proteins,carbohydrates, lipids, hyaluronic acid, laminin, fibronectin,pluripotent mesenchymal stem cells (MSC) and other complex growthfactors that are essential for fetal growth and development. Inparticular, amnion has a complete lack of surface antigens, thus it doesnot induce an immune response when implanted into a ‘foreign’ body,which is in contrast to most other allograft implants. Amnion alsomarkedly suppresses the expression of the pro-inflammatory cytokines,IL-1α and IL-1β (Solomon et al., 2001, Br. J. Ophthalmol. 85 (4):444-9)and produces natural inhibitors of matrix metalloproteases (MMPs)expressed by infiltrating polymorphonuclear cells and macrophages (Haoet al., 2000, Cornea, 19 (3):348-52; Kim et al., 2000, Exp. Eye Res. 70(3):329-37). Amnion also down-regulates TGF-β and its receptorexpression by fibroblasts leading to the ability to modulate the healingof a wound by promoting tissue reconstruction. Furthermore, amnion has abroad spectrum of antimicrobial activity against bacteria, fungi,protozoa, and viruses for reduced risk of post-operative infection.

Amnion derived tissues are therefore immune-privileged and ideallysuited for regenerative medicine applications.

A “kit” is an assembly of parts, materials, and compositions of matterpackaged together to facilitate a treatment. Kits commonly compriseinstructions for the use of the parts, materials and compositions.

Turning now to FIG. 1, a kit embodying features of the presentinvention, generally designated by the numeral 11 is depicted. Kit 11has the following major elements: a first vial 15, a second vial 17, acontainer for a tissue wrap 21, a syringe 23, and instructions 25. Thekit 11 is held in suitable packaging, as depicted, a box 27. Suitablepackaging may comprise any means for holding the collection of parts,materials and compositions. For example, without limitation, bags,wraps, containers, ties and the like. The kit 11 for ocular treatmentsmay comprise fibrin glue [not shown] for attaching the tissue wrap 23 tothe eye.

The first vial 15 contains a dried particulate mixture of mechanicallydecellularized amnion obtained from one or more animals compatible witha subject animal that upon of reconstitution forms a reconstitutedmedicament. The kit 11 may contain a vial containing such liquid forreconstitution [not shown] or the liquid for reconstitution may bederived from other sources.

The second vial contains fetal cells obtained from one or more animalscompatible with a subject animal and compatible with a particulatemixture in the first vial 15. The fetal cells are applied in cooperationor concurrently with the reconstituted medicament or form a combinedmedicament comprising a particulate cellular suspension medicament foradministration to the subject animal to promote healing. In forming acombined medicament, the dried particulate mixture of the first vial 15is reconstituted with or combined with the fetal cells of the secondvial 17, supplemented as needed with further liquid for reconstitution.For example, the dried particulate mixture can be reconstituted bysuspension in a solution of 50% solution for reconstitution, such asPlasmaLyte ATM, and 50% amniotic fluid containing fetal cells.

The combined medicament is injected into or around the site of injurywith syringe 23.

The container for a tissue wrap 21 contains a tissue wrap derived fromamnion tissue obtained from one or more animals compatible with asubject animal. The tissue wrap comprises amnion tissue constructed andarranged for placement in juxtaposition with the site of injury. Thetissue wrap is applied in cooperation or concurrently with thereconstituted medicament or a combined medicament comprising aparticulate cellular suspension medicament for administration to thesubject animal to promote healing.

Turning now to FIG. 2 the container for tissue wrap 21 is depicted as atransparent bag through which the tissue wrap designated by numeral 31can be seen. As seen in FIG. 3, tissue wrap 31 is formed by affixingamnion tissue to a first support 33 on one side of the amnion and asecond support on the other side of the amnion [not shown] and airdrying the tissue for thirty minutes to three hours or more, and, mostpreferably, for about one hour. The supports, of which first support 33is depicted, maintain the shape of the tissue during the drying process.The supports are preferably removed prior to placement of the tissue incontainer 21.

Tissue wrap 31 is removed from the container and placed over the site ofinjury.

Returning now to FIG. 1, the dried particulate mixture is obtained fromthe mechanical decellularization or cryofractionation of about 1.5 10⁻²cm³ to 5 10⁻² cm³ of amnion/mL of reconstituted medicament and includeparticles greater than 20-100 microns in diameter. Upon reconstitutionof the dried particulate mixture by suspension in a solution of amnioticfluid and/or PlasmaLyte A™, the reconstituted medicament can beadministered to the subject animal to promote the healing of superficialwounds.

A reconstituted medicament containing particles less than about 100microns is suitable for treatment of eye injuries. The kit 11 for thetreatment of eye injuries comprises a dropper for administration.

The dried particulate mixture obtained from the cryofractionationcomprises about 1.5×10⁻² cm³ to about 5×10⁻² cm³ of amnion/mL ofreconstituted medicament.

The fetal cells can comprise amniotic fluid cells and the particulatematter can be filtered to contain particles that are less than 100microns in diameter. The fetal cells can have a concentration from 10³to 10²⁰/mL mesyschimal and/or epithelial stem cells. In anotherembodiment, fetal cells can have a concentration of 10³ to 10¹²/mL. Inanother embodiment, fetal cells can have a concentration of 10⁴ to10¹²/mL. In another embodiment, fetal cells can have a concentration of10⁴ to 10¹¹/mL. In another embodiment, fetal cells can have aconcentration of 10⁴ to 10¹⁰/mL. In another embodiment, fetal cells canhave a concentration of 10⁴ to 10⁹/mL. In another embodiment, fetalcells can have a concentration of 10⁴ to 10⁸/mL. In another embodiment,fetal cells can have a concentration of 10⁴ to 10⁷/mL. In anotherembodiment, fetal cells can have a concentration of 10⁴ to 10⁶/mL. Inanother embodiment, fetal cells can have a concentration of 10³ to10⁶/mL. In another embodiment, fetal cells can have a concentration of10³ to 10⁷/mL.

In one example, the fetal cells can have a concentration of about0.8×10⁶ to 1.2×10⁶ cells/mL of the particulate cellular suspension. Thedried particulate mixture can contain particles obtained from thecryofractionation of from about 1.5×10⁻² cm³ to about 5×10⁻² cm³ amnionper mL of the particulate cellular suspension. In one example, theparticulate cellular suspension comprises a particulate matter obtainedfrom the cryofractionation about 1.5×10⁻² cm³ to about 5×10⁻² cm³ amnionfor every 10⁶ plus or minus 2×10⁵ amnion fluid cells.

Embodiments of the present invention are well suited for the treatmentof open wounds, superficial and deep including such wounds that expose amovable body element of the animal's musculoskeletal system, forexample, a ligament or tendon. Such wounds can include ligaments andtendons that are lacerated.

For example, in the event the animal is a horse and the injury maycomprise chondral and fascial pathologies, tendon and ligamentpathologies, surface reconstruction or articular joint pathologies. Thechondral and fascial pathologies can include, for example, laminitis.The tendon and ligament pathologies can include, for example,tendonitis, sport-induced superficial digital flexor tendon (SDFT) orsuspensory ligament (SL) lesions. The articular joint pathologies caninclude, for example, navicular disease, ringbone or arthritis. The siteof injury can also be an open wound that, in one example, is able toheal in about one to two weeks. In another example, healing of the siteof injury reduces the risk of recurrence. In one example, the risk ofrecurrence of the injury can be reduced by about 5 fold.

Features of the present invention are further described with respect tothe following Examples. These examples feature equine subjects andmaterials. However, materials derived from other animals species, forexample, without limitation, canine, feline, bovine, porcine and otheranimal species materials and subjects can be prepared in a similarmanner as outlined below. The equine materials have been utilized withavian, reptilian and other animal groups.

EXAMPLES Example 1: Amniotic Material Processing

This procedure defines the aseptic collection of amniotic material(amnion and amniotic fluid) for injection at the site of an injury.

Amnion Tissue

The amnion container was picked up and sampled for Bioburden. The amnionwas aseptically transferred into the sterile field (laminar flow hood).The amnion transport packaging (previously disinfected, i.e. with 70%ETOH) was opened.

A 50 mL sample of the Amnion Transport Solution was asepticallytransferred into a 30 to 60 mL conical tube for pre-processing bioburdentesting. The vial was labeled with sample description, batch number,date and time and placed in a designated refrigerator.

(1) Amnion Preparation

The amnion from the incoming container was transferred intoapproximately 200 mL of Plasma Lyte-A in a sterile bioassay dish whereit was gently rinsed. A piece of amnion was then spread evenly on asterile cutting board carefully avoiding any overlaps. A record was madeof the amnion preparation start time. Sterile gauze or laps were used toremove any remaining debris/blood from the surface of the amnion. Theamnion was inverted and the surface of the opposite site was similarlywashed. Any chorion was removed by blunt dissection to separate it fromthe amnion. After washing and cleaning, the amnion pieces were returnedto the bioassay dish containing Plasma Lyte-A. Using a sterilescissors/scalpel, the amnion was cut into 2 to 10 sections. Theapproximate area of each piece (50-450 cm²) was measured and recordedusing a sterile stainless steel ruler.

The cleaned pieces of the amnion were placed back on the sterile cuttingboard and the amnion was spread out on the board taking care to notoverlap. A sterile nylon mesh was placed over the surface of the amnionagain taking care to overlap them. The amnion and mesh were then placedonto a sterile drying rack and allowed to air dry for a minimum of onehour. Start and stop times for drying were recorded.

(2) Amniotic Fluid

(a) Amnion Fluid Preparation

A large sterile pan was first placed into the sterile field (laminarflow hood) and filled with cold packs from a −80° C. freezer. Theaspiration containers with the amniotic fluid were disinfected with 70%ethanol (ETOH), inspected for integrity and placed on the cold packs inthe laminar flow hood. The source of the amniotic fluid was confirmed bylooking at the Donor animal ID number. The 2.0 mL of amniotic fluid wasthen aseptically pipeted into a 2 mL sterile microcentrifuge tube forbioburden testing. The vial was labeled with the sample number, batchnumber, date and time and placed in the designated refrigerator.

Using a 50 mL sterile disposable serological pipette all the remainingamniotic fluid was transferred into 1 liter sterile disposable bottlesand placed on the cold pack. The total volume of the amniotic fluid andthe color were recorded.

(3) Amniotic Fluid Cell Count and Determination of the Number of ViableCells

Each amniotic fluid bottle was gently mixed and 1.0 mL of the fluid wascollected using a 1 mL micropipette and transferred into a 2 mLEppendorf microcentrifuge tube. A total of 50 μL of cell suspension wasthen added to 50 μL of trypan blue (0.4%) in an Eppendorfmicrocentrifuge tube and vortexed for 5 seconds. The sample was placedon a rack for 5 minutes.

A Neubauer chamber (hemocytometer) was rinsed with distilled water, andthen sprayed with ETOH 70% and wiped clean and dried with paper towels.A cover slip was placed on the top of the micro-grids of the chamber.The trypan blue-cell suspension was gently mixed and used to fill bothsides of the hemocytometer with 10 μL by capillary action. The cellswere allowed to settle down for at least 30 seconds. The hemocytometerwas placed under the microscope and all cells in the four 1 mm cornersquares and one 1 mm center square were counted. For accuracy the totalnumber of cells counted was greater than 100. The cells were re-countedif >10% of the cells appeared clustered, by vigorously pipetting in theoriginal cell suspension as well as in the trypan blue cell suspensionmixture. Using a double cell counter, the number of viable andnon-viable cells was determined. The cells in both chambers were countedand an average was calculated. For the trypan blue test, live cells didnot take up the dye, whereas dead (non-viable) cells did. Thusnon-viable cells stained blue and viable cells remained opaque. “Ghost”cells, which appeared as flattened pale blue cells were not counted.

The number of cells was determined as follows: Each square represented atotal volume of 0.1 mm³ or 1×10⁻⁴ mL (0.1 mm depth×1 mm width×1 mmheight=0.1 mm³). The number of cells per mL was then deduced from theaverage viable cell count per 1 mm square×2×10⁴. The total cell numberwas therefore equal to the number of cells per mL multiplied by theoriginal volume of sample fluid. The cells were then diluted to thedesired concentration.

Example 2: Aseptic Processing of the Amniotic Material

(1) Aseptic Cryofractionation of Amnion

After at least one hour, the amnion was removed from the drying rack andtransferred into the milling chambers having an impactor. The millingchambers were placed into the Cryomill and cryofractionated using thefollowing settings:

Number of Cycles: 4

Frequency 1/s: 10 CPS_(—)

Precooling Time: 10 minutes

Grinding Time: 4 minutes

Intermediate Cooling: 3 minutes

Once grinding was complete, the milling chambers were allowed to warm toroom temperature for approximately two hours. The start and stop timeswere recorded.

Approximately 50 mL of the amnion suspension solution was dispensed intoeach milling chamber. The inside milling chamber and the impactor wererinsed with the solution multiple times until the ground amnion (driedparticulate mixture) was re-suspended and collected in the bottom of thechamber. The impactor was removed using the magnet pen. Thecryofractionated amnion solution was then transferred to the amnionsuspension container and placed on cold packs in the sterile field anddiluted to the desired amount.

(2) Aseptic Processing of Amniotic Fluid

The amniotic fluid was aliquoted evenly into 50 mL sterile centrifugetubes and centrifuged at 200-400×g (1100-1600 rpm) for approximately5-10 minutes at room temperature. The supernatant was then removed fromeach tube using a 25 mL sterile serological pipette. The amniotic liquidwas kept in new container and the pellet was re-suspended in PlasmaLyte-A™ to a total volume of 25 mL in each tube. The re-suspended cellsin any two different tubes were vortexed for approximately 3 to 5 secand consolidated into a single tube prior to centrifugation at 200-400×g(1100-1600 rpm) for approximately 5-10 minutes at ambient temperature.The preceding steps were repeated as necessary.

The supernatant from each tube was removed using a sterile pipette andthe pellet was again re-suspended in a cell suspension solution(amniotic fluid and/or an isotononic solution, e.g., PlasmaLyte A™) tobring the volume in each tube to about 10 mL and vortexed forapproximately 3 to 5 seconds. A 1 mL aliquot was removed and the cellcount and viability was determined using the above-described trypan bluetest.

If red blood cells were present in the amniotic fluid cell suspension,they were removed using a RBC Lysing Solution. A 10× concentration wasprepared as follows: NH₄Cl (ammonium chloride)=8.02 gm+NaHCO₃ (sodiumbicarbonate)=0.84 gm adjusted to a total volume of 100 mL with Milliporefiltered water. 10 mL of the 10× concentrate was added to 90 mLMillipore filtered water and refrigerated until use. The amount neededof Erythrolysis solution (15 mL per tube centrifuged) was removed fromthe refrigerator and kept for a period of 0.5 hours in the stabilizationincubator. After centrifuging the amniotic fluid at 400×g for 10minutes, the supernatant was removed and the pellet was re-suspended inErythrolysis solution (minimum of 50 mL per tube). The contents of allthe tubes were consolidated into one tube that was rocked for ˜10minutes at room temperature until the liquid was clear red. The cellswere again centrifuged for 5 minutes at 250 to 400×g. The supernatantwas decanted. The pellet was washed with 50 mL of PBS or PlasmaLyte A™before centrifugation again for 10 minutes at 250 to 400×g. The washingof the pellet was repeated as needed. The amniotic fluid cells were thenfiltered through a 100 μm cell strainer, and re-suspended in PBS orPlasmaLyte ATM. The cells were again centrifuged for 10 minutes at 250to 400×g. The supernatant was decanted and the pellet was left in the 50mL conical centrifuge tube.

(3) Procedure for Cryopreservation of Cryofractionated Amnion withAmnion Fluid Cells

Appropriate size cryovials that were previously labeled and theircorresponding size of CoolCell™ freezer (CCF) racks were placed in thehood. CryoStor 10®, the Cell Suspension Solution and the AmnionSuspension Solution were also placed on cold packs in the hood. Cryostor10™ is commercially available from Biolife Solutions.

The cell suspension solution and the amnion suspension solution werethen combined into the cell suspension solution container. Using 50 mLserological pipettes, the solutions were homogenized several times. Thecontainer was again placed on the cold packs on the sterile field. Emptycryovials were placed in the CCF racks on cold packs and their caps wereremoved inside the hood (sterile field). The mix of cell/amnionsuspension solution was pipeted into an empty Amnion Suspensioncontainer and a same volume of CryoStor 10™ was added and homogenizedbefore being placed on the cold packs. A 50 mL pipette Combitip wasfitted on to a repeat pipetor set to dispensing mode. The fill volumewas adjusted and 50 mL of the Cell/Amnion solution was aspirated andthen adjusted to the desired dispense volume (1 or 2 mL). The cryovialsin the CCF were then filled and the vial caps were replaced securely andthe rack of filled vials was placed on cold packs for QC inspection.

Cell freezing was achieved by cooling the cells at a cooling rate of 1°C. per minute from 4° C. to −80° C., using a passive coolingcontrolled-rate freezer CoolCell™ (commercially available fromBiocision.)

The cell suspension in cryoprotective freezing medium was aliquoted intoeach of the cryovials and the cells were gently mixed to maintain ahomogeneous cell suspension. The solid core of the CoolCell (black ring)at room temperature was seated in the bottom of the central cavity andthe vials containing the cell suspension were placed in each well. Thelid of the CoolCell™ was fully sealed and the Coolcell™ was placed intoa −80° C. freezer for at least 4 hours prior to transfer on dry ice tolong term storage. Cell viability and QA/QC were evaluated by thawingone vial after short term storage.

Example 3: Amniotic Tissue Wrap Preparation

After confirming the amniotic tissue source and donor mare ID andrecording the time of receipt, the amnion transport packaging(previously disinfected, i.e. with 70% ethanol, methanol, etc.) wasaseptically transferred into the sterile field (a laminar flow hood). Asample of the Amnion Transport Solution was first transferred into a 50mL conical tube for Bioburden testing. The vial was then labeled withsample description, batch number, date and time and placed in designatedrefrigerator.

(1) Amniotic Membrane Wrap Preparation

Saline was aseptically added into a second receiving pan in the sterilefield (i.e. laminar flow hood) and the amnion tissue was taken fromincoming receiving pan to the second receiving pan containing thesterile saline. Any remaining blood was rinsed with sterile saline.After documenting the amnion preparation start time, sterile gauze orlaps was used to remove any remaining debris/blood from the surface ofthe amnion. The amnion was then inverted and the other side was rinsedand washed. Any remaining chorion was removed by blunt dissection toseparate it from the amnion. The amnion was kept wet with sterilesaline. The tissue was blocked off by cutting away any stringy ends andchecked for holes or tears. After repositioning the amnion on thecutting board with the chorion side up, the approximate area of eachpiece of amnion was measured and recorded in cm² using a sterilestainless steel ruler.

The amnion (chorion side up) was covered with a sterile mesh (e.g., anylon mesh) wetted with sterile saline. For the purpose of thisdiscussion, there is a first steel mesh which is denoted by a firstvisible indicia, the color white, and a second steel mesh, to bediscussed below, which is denoted by a second visible indicia, the colorblue. Any reference to the colors white or blue are directed to thesevisible indicia. Those skilled in the art will recognize that suchvisible indicia is matter of choice. The mesh was cut to size and wasallowed to slightly overlap the amnion. The mesh-covered amnion was thengently lifted, turned over and placed back on the cutting board. Cautionwas used as to not disturb the mesh/amnion interface. The newly exposedside of the amnion was wiped with sterile wipes or gauze pads to removeany remaining blood or small tissue particles. The newly exposed side ofthe amnion was covered with a second steel mesh, BLUE sterile mesh(e.g., a nylon mesh) wetted with sterile saline. The mesh was cut tosize and was allowed to slightly overlap the amnion.

The amnion tissue sandwiched between the white and blue mesh was placedon the drying rack where the amnion tissue was allowed to dry for 60 to90 minutes at ambient temperature (65° to 70° F.). Caution was used asto not disturb the mesh/amnion interface. The amnion was kept unfoldedand as flat as possible during this step. Additional drying racks wereused as needed. The total drying time was recorded. The dried tissue wasthen removed from the rack(s) and laid flat on the cutting board, WHITEside up. The WHITE mesh was carefully removed from the entire sheet ofamnion which was checked for holes or tears. With the BLUE mesh side upon the cutting board, each section was cut using a scalpel or rotarycutting blade and the sizes and surface areas were recorded as 5×5 cm,10×10 cm, round 15 mm diameter and round 22 mm diameter. Those skilledin the art will recognize that the sections can be cut and sized to fitparticular needs and these sizes and shapes are only exemplary.

(2) Amniotic Membrane Wrap Pre-Packing

Pouches, Sealing Test and Labeling

The sterile field was set up for packaging including a sealer forpacking pouches (Sealer settings: Temperature=177±9° C. (350±15° F.)).Three (3) empty pouches were sealed for visual inspection and retentionand then labeled. Packing pouches are available from niumerous vendors.One suitable pouch is sold under the trandemark KAPAK™.

Amniotic Membrane Wrap Packing

Pouches were transferred onto the sterile field (previously disinfectedlaminar flow hood). Using sterile forceps, each individual tissuemembrane was inserted into the inner pouch. Large membranes were foldedif necessary. The pouches were sealed with the dried amnion tissue on amesh (see, for example, FIGS. 2 and 3) and inspected for a broken seal,impurities, and defects. Upon passing the inspection, the pouches werelabeled with date and packaged in large pouches according to size andstored in the refrigerator or at room temperature. The donor ID, size,date, time, and initials were documented.

Example 4: Implantation of Cryofractionated Amnion and Isolated AmnioticFluid Cells

(1) Thawing Vials

A container was ¾ filled with hot tap water with a thermometer and coldwater was added until a temperature of 37° C. was reached. A vial ofcryofractionated amnion with amniotic fluid cells was taken from the−80° C. freezer. Holding the cap, the vial was partial immersed in thewater bath for approximately 2 to 3 minutes with gentle agitation untilthe contents were melted. The vial was removed from the water bath andthe exterior was wiped with sterile gauze saturated with 70% ethanol.The thawed contents were then ready for immediate use.

(2) Animal Restraint

A horse with an injury was adequately restrained and sedated oranaesthetized before starting the procedure according to theveterinarian's directions. Appropriate sedation or anesthesia causedminimal distress to the patient and prevented any long lastingdiscomfort. Use of nose twitch was optionally required. In the case ofyoung adults or unbroken horses, short-acting general anesthesia wasrequired.

(3) Implantation Procedure

The site for the product implantation was processed as a surgicallyprepared area. After cleaning or clipping of any gross contamination,such as dust or mud, the complete the area was scrubbed withchlorhexidine for 5 to 7 minutes and then wiped down with alcohol swabs.Before proceeding, the area was cleaned until the alcohol swabs used onthe scrubbed area were dirt-free. Systemic antibiotics were administeredprior to starting the procedure. However, in some instances, systemicantibiotics are not necessary.

The contents of the 2 mL vial were split into 3 to 4 doses (0.50 to 0.67mL) and loaded into syringes (for example, 1 mL syringes) with sterilehypodermic needles (for example 22 gauge, 1.5 inch needles). Theappropriate leg was resting or held up and restrained as needed. Forinjuries to a tendon, ligament or muscle the cryofractionated amnion andamniotic fluid cell mix was injected in close proximity to the lesionwithout penetrating the actual tendon, ligament or muscle. For injuriesto articulation (i.e., joints) the cellular particulate mixture wasinjected either peri-articularly or intra-articularly. After injection,the site was bandaged with sterile swabs and adhesive bandage.

Example 5: Implantation of Cryofractionated Amnion and Isolated AmnioticFluid Cells in Combination with the Amnion Tissue Wrap

(1) Animal Restrain

A horse was adequately restrained as described in Example 4.

(2) Materials

A kit comprising sterile hypodermic needles (18 and 23 gauge), syringes(3 to 5 mL), vials of thawed cryofractionated amnion and amniotic fetalcells as well as packaged amnion tissue wrap of the appropriate sizewere assembled (see FIG. 1) together with clippers, sterile gloves, 1%lidocaine, saline and sedatives and/or anesthetics.

(3) Wound Surface Preparation

A horse was treated at a veterinary clinic a severe leg severe injurycaused by barbed wire. The wound was initially pre-cleaned to removedirt, scrubbed with chlorhexidine for 5 to 7 minutes and then wiped downwith alcohol swabs.

(4) Sharp surgical debridement:

The veterinarian aggressively debrided the wound. This step created arelatively clean wound bed and generated an inflammatory signal thatinduced the migration and proliferation of stem cells and growth factors(from the amniotic material and horse's own immune system). Systemicantibiotics were administered prior to starting the procedure.

(5) Treatment of an Open Wound with Amniotic Tissue Wrap

The veterinarian determined the amount of amniotic material needed totreat the wound by measuring the maximal length, width and depth of thewound. Amniotic tissue wrap of the appropriate size was selected andapplied on the wound. Treatment of an open wound with Amniotic tissuewrap and a cellular suspension of cryofractionated amnion and isolatedamnion fluid cells.

After the application of the amnion tissue wrap as described above, theapproximate volume of the open wound volume was estimated and the amountof cryofractionated amnion and isolated amnion fluid cells to beinjected were determined in accordance with guidelines shown in Table 3.

TABLE 3 Amount of cryofractionated amnion and isolated amnion fluidcells to be injected as a function of wound volume. Amount ofcryofractionated amnion/ Wound Area amnion fluid cell suspension to use<12.5 cm² 1.0 mL >12.5 cm² but <25 cm² 1.5 mL   >25 cm² 2.0 mL

After debridement, the cryofractionated amnion/amnion fluid cellsuspension was injected into the pen-wound skin that appeared normal intexture, turgor, and free of infection using a 23-gauge needle on a 3 mLsyringe.

The cell suspension was injected at a distance of approximately 5 mmfrom the wound edges and 10 to 15 mm deep at the 3-, 6-, and 9- and12-o'clock positions (directly into the superficial fascia and thesubcutaneous tissue of the wound), aiming the needle parallel to thewound margin at each location. To minimize discomfort and to ensure anadequate spread of the amniotic material, the cryofractionatedamnion/amnion fluid cell suspension was mixed with 1% plain lidocaine ina 1:1 ratio. After the completion of the procedure, the surgical sitewas dressed with a nonporous dressing followed by application of anonstick dressing and a dry sterile dressing of gauze. Five to sevendays after implant of the amnion allograft, the wound was redressed, andstandard wound care consisting of saline wet to dry sterile gauzedressing was resumed. If the wound proved hard to heal, additionalapplications of the amnion tissue wrap and/or cryofractionatedamnion/amnion fluid cell suspension were made using the same techniqueat 14- to 21-day intervals until the wound healed. Concomitant therapieswere given i.e. wound offloading, and/or edema control as recommended bythe veterinarian.

(6) Healing of Wound

The wound was assessed and showed striking improvement. Generally,treatment shows 2.5 times the rate of healing.

Example 6: Treatment of Eye Injuries with the Amnion Tissue Wrap

(1) Animal Restrain

A horse was adequately restrained as described in Example 4.

(2) Eye Surface Preparation

Corneal ulcers are the most common eye problem in horses. A veterinarianlocated the foreign material and determined the wound severity (size ofthe corneal lesion) using visual inspection or fluorescein dye tohighlight any subtle corneal defect or scratch. The area around the eyewas cleaned to remove debris or excessive liquid using sterile gauze.

(3) Application of the Amniotic Tissue Wrap

An appropriate size of amniotic tissue wrap (ie. 15 or 22 mm diameterdiscs) was selected. The dehydrated amniotic tissue wrap was positionedover the tip of the index finger and applied directly to the eye surfacewhile opening the eye lids with the other hand (if needed). The membraneattached to the eye surface by hydrostatic tension (glue or sutures maybe used to assist to insure placement in challenging environments.).

Example 7: Treatment of Eye Injuries with a Suspension ofCryofractionated Amnion

(1) Animal Restrain

A horse was adequately restrained as described in Example 4.

(2) Eye Surface Preparation

The eye surface and surrounding area was prepared as described inExample 6.

(3) Application of Eye Drops of Cryofractionated Amnion

Eye drops were given either alone or in conjunction with the amniotictissue wrap application described in Example 6. Vials ofcryofractionated amnion suspended in saline (ie. PlasmaLyte A™ and/orAmniotic Fluid). were thawed (36 to 37° C. or 97 to 98° F. water bath)for at least 5 min. The vial was gently rotated upside down to mixsolution. 2 drops of the suspension were applied to the injured eyetwice a day (4 drops total per day) over the membrane or ocular surface.Treatment lasted for a week or until the suspension was used up. Thesuspension was pH balanced to prevent any irritation to the eye.

The invention is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the invention inaddition to those described will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

Any patent, patent application, publication, or other disclosurematerial identified in the specification is hereby incorporated byreference in its entirety and for all purposes to the same extent as ifeach such individual reference (e.g., patent, patent application,publication, or other disclosure material) was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes. Any material, or portion thereof, that is said to beincorporated by reference herein, but which conflicts with existingdefinitions, statements, or other disclosure material set forth herein,is only incorporated by reference to the extent that no conflict arisesbetween that incorporated by reference material and the presentdisclosure material.

Other embodiments are within the following claims.

1.-97. (canceled)
 98. An article of manufacture comprising: (i) a driedparticulate mixture of mechanically decellularized fetal tissue obtainedfrom one or more animals compatible with a subject animal, the driedparticulate mixture is capable of reconstitution to form a reconstitutedmedicament for administration to the subject animal to promote healing;and (ii) fetal cells obtained from one or more animals that arecompatible with the dried particulate mixture, wherein combining thedried particulate mixture and the fetal cells forms a medicamentcomprising a particulate cellular suspension for administration to thesubject animal to promote healing, wherein the subject animal is anon-human animal.
 99. The article of manufacture of claim 98, whereinthe fetal tissue and/or fetal cells comprises amnion or chorion. 100.The article of manufacture of claim 98, wherein the dried particulatemixture is obtained from the mechanical decellularization of about1.5-5×10² cm³ of amnion/mL of reconstituted medicament.
 101. The articleof manufacture of claim 98, wherein the dried particulate mixturecomprises particles greater than about 100 microns in diameter.
 102. Thearticle of manufacture of claim 98, wherein the dried particulatemixture contains particles of less than about 100 microns in diameter.103. The article of manufacture of claim 98, wherein the particulatecellular suspension comprises about 0.8-1.2×10⁶ amnion fluid fetalcells/mL of medicament.
 104. The article of manufacture of claim 98,further comprising an air dried fetal tissue wrap obtained from one ormore animals compatible with a subject animal, the fetal tissue wrapconstructed and arranged for placement in juxtaposition with a site ofinjury to promote healing.
 105. The article of manufacture of claim 98,wherein the fetal tissue comprises amnion and/or chorion.
 106. Thearticle of manufacture of claim 104, wherein the wrap is constructed andarranged for placing on the site of at least one of an eye injury or acorneal ulcer.
 107. The article of manufacture of claim 98, wherein thehealing occurs at a site of injury that exposes a movable body elementof the animal's musculoskeletal system, wherein the movable body elementis a muscle, joint, ligament, or tendon, wherein the injury comprises alaceration.
 108. The article of manufacture of claim 107, wherein thehealing is at a site of injury caused by a condition selected from thegroup consisting of chondral and fascial pathologies, tendon andligament pathologies, surface reconstruction and articular joint path109. A method for treating an injury in a subject animal, comprising thesteps of: administering the article of manufacture of claim 98 to theperiphery of an injury of a non-human animal, wherein the administeringthe particulate cellular suspension promotes healing of the injury,wherein the administering comprises any one or more of an intravenousinjection, an intra-articular injection, a subcutaneous injection, asuperficial injection or a nebulization.
 110. The method of claim 109,further comprising the step of applying an air dried fetal tissue wrapto the site of the injury.
 111. The method of claim 109, wherein theinjury is debrided prior to injecting.
 112. The method of claim 109,wherein the injury is an eye injury.
 113. A method, comprising: (i)obtaining animal fetal tissue; and (ii) decellularizing at least aportion of the animal fetal tissue to obtain a particulate mixture. 114.The method of claim 113, further comprising reconstituting theparticulate mixture through adding a biocompatible solution to theparticulate mixture, wherein adding the biocompatible solution creates areconstituted particulate mixture comprising fetal cells, wherein thereconstituted particulate mixture is suitable for treating injuries tocompatible injured animals.
 115. The method of claim of claim 114,further comprising; (i) filtering the reconstituted particulate mixtureto increase the concentration of fetal cells; and (ii) injecting theparticulate mixture into an injured animal, wherein the injection ismade to a periphery of an injury of the injured animal.
 116. The methodof claim 115, wherein decellularizing at least a portion of the animalfetal tissue comprises obtaining extracellular matrix through mechanicaldecellularization comprising cryofractionation.
 117. The method of claim113, further comprising obtaining animal amnion fluid.